Our goal is to understand the molecular mechanisms controlling assembly and disassembly of actin filaments during cellular locomotion in terms of the rates of specific reactions. We will use a combination of biochemical, genetic and cellular experiments, largely in fission yeast, to test how cells initiate new actin filaments and disassemble aged actin filaments. We will focus on actin, Arp2/3 complex, WASp/Scar proteins, capping protein, ADF/cofilins and profilin, all-important components of the system. We will determine all of the rate and equilibrium constants required to explain the thermodynamics of actin filament treadmilling. To learn how cells activate Arp2/3 complex we will used novel spectroscopic assays to analyze the interactions of Arp2/3 complex with each of its activators (actin monomers, actin filaments, WASp including variants with disease-causing mutations) and test molecular and cellular mechanisms through mutagenesis. We will determine the effect of the nucleotides associated with the mother filament and with Arp2/3 complex on branch formation as well as the parameters that control the stability and lifetime of branches. We will create and characterize inhibitors of branching by Arp2/3 complex to test the dependence of actin-based processes in cells on Arp2/3 complex. We will characterize the effects of VASP, tropomyosin and PIP2 on actin filament capping and annealing. To learn how cells control actin filament disassembly and subunit recycling, we will characterize actin filament severing and depolymerization by [unreadable] ADF/cofilin proteins characterize the competition of phosphate and ADF/cofilin for ADP-actin filaments and determine the structure of ADF/cofilin bound to actin. We will attempt to reconstitute a recycling actin motility system from purified proteins and analyze the dynamics of individual filaments by total internal reflection fluorescence microscopy. We will determine the mechanism of actin filament nucleation by formin proteins and use suppressor analysis to verify that Cdc12 is the (exclusive) poly-L-proline ligand for profilin in S. pombe. [unreadable] [unreadable]